Efforts to increase the sensitivity of nucleic acid hybridization reactions have been a recurrent feature of the molecular biology landscape for the past 30 years. Among other advantages, increases in sensitivity serve to decrease the amount of sample, probe, and other reagents required to detect a nucleic acid target, of particular importance when a biological sample occurs in limiting amount.
Early efforts were directed to increasing detection sensitivity in hybridization reactions performed using radiolabeled probes and included, for example, the development of methods for increasing the specific activity of radiolabeled probes, such as random primed hexamer labeling, and of methods that increased detection sensitivity itself, such as the use of rare earth intensifying screens at subzero film exposure temperatures. The later introduction of phosphorimaging techniques made possible the electronic amplification of radioactive signals.
Other efforts, not limited to radioisotopic labeling, have included alterations in probe composition, such as use of single-stranded RNA transcripts, and more recently, the introduction of peptide nucleic acids, respectively reducing competition and increasing the stability of the resulting probe:target duplex.
Enzymatic amplification of signal has also been used to increase sensitivity, with the enzyme variously catalyzing deposition of optically detectable colored product or, in the case of enhanced chemiluminescence, catalyzing the production of light.
More recently, improvements in sensitivity have been attained by physical amplification of target, either before or during detection. The advantages of target amplification—including the ability to start with picomolar amounts of starting material, the ability to decrease the complexity of a sample by specific amplification of a desired sequence, and the ability to monitor amplification in real time—have led to a variety of amplification protocols, such as polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), self-sustained sequence recognition (3SR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), rolling circle amplification (RCA), and strand displacement amplification (SDA).
Yet all of these approaches have disadvantages.
For example, there is a physical limit to the number of labels that can be incorporated into a probe of given length. Enzymatic methods for amplifying signal require careful attention to technique, and may require expensive reagents. Physical amplification of target suffers from nonspecific amplification; where mispriming occurs early in the amplification reaction, the erroneous template may be amplified to nearly as great an extent as the desired target. Additionally, methods of target amplification that require thermocycling require additional apparatus, and may preclude real-time detection when the sensitivity of the detection means is itself affected by changes in temperature.
Hammond et al., U.S. Pat. No. 6,255,051, taking a different approach, recently described a “recursive cascade” of strand displacement reactions with gain of signal at each individual displacement reaction, which is said to lead to signal amplification. However, the scheme requires at least seven engineered probe molecules for each target to be detected.
There thus exists a continuing need in the art for methods, devices, compositions and kits for amplifying the signal in nucleic acid hybridization reactions, and particularly for nonenzymatic, isothermal methods for amplifying the signal without physical amplification of target.